Abstract

Mammalian target of rapamycin (mTOR) constitutes a nodal point of a signaling network that regulates cell growth and proliferation in response to various environmental cues ranging from growth factor stimulation to nutrients to stress. Whether mTOR is also affected by cholesterol homeostasis, however, has remained unknown. We report that blockade of cholesterol trafficking through lysosome by a newly identified inhibitor of angiogenesis, itraconazole, leads to inhibition of mTOR activity in endothelial cells. Inhibition of mTOR by itraconazole but not rapamycin can be partially restored by extracellular cholesterol delivered by cyclodextrin. Moreover, other known inhibitors of endosomal/lysosomal cholesterol trafficking as well as siRNA knockdown of Niemann–Pick disease type C (NPC) 1 and NPC2 also cause inhibition of mTOR in endothelial cells. In addition, both the accumulation of cholesterol in the lysosome and inhibition of mTOR caused by itraconazole can be reversed by thapsigarin. These observations suggest that mTOR is likely to be involved in sensing membrane sterol concentrations in endothelial cells, and the cholesterol trafficking pathway is a promising target for the discovery of inhibitors of angiogenesis.

The mammalian target of rapamycin (mTOR) pathway plays a key role in sensing and integrating multiple environmental signals to regulate cell growth and proliferation (1, 2). mTOR exists in two distinct complexes in mammalian cells: mTOR complex (mTORC) 1 and mTORC2. Although mTORC1 is involved in regulating translation, ribosomal biogenesis, and autophagy mediated, in part, by activation of p70 S6 kinase (p70S6K) (3, 4) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) (5, 6), mTORC2 has been shown to affect cellular cytoskeleton as well as Akt phosphorylation (7). Among the upstream signals that are known to affect the mTOR pathway are growth factors, nutrients such as amino acids, cellular energy status, and a variety of environmental stresses.

Cholesterol constitutes a unique type of cellular building block. It is responsible for conferring the fluidity and impermeability to cellular membranes, and hence the existence of individual cells. It is also known to play an essential role in signal transduction as an essential component of lipid rafts (8). There are two sources of cholesterol: those that are synthesized in the endoplasmic reticulum (ER) (9) and those that are acquired from extracellular space via LDL receptor-mediated endocytosis (10). Both pools of cholesterol require proper intracellular transport to reach their final destinations. Given its key roles in membrane structure and function and in signal transduction, cholesterol homeostasis is under tight control. Cells employ at least two sensor proteins, Scap and 3-hydroxy-3-methylglutaryl CoA reductase, to monitor the levels of membrane sterols and to regulate cholesterol biosynthesis (11). How membrane cholesterol levels regulate cell proliferation, however, has remained unknown.

In a previous study, we screened a library of known drugs, the Johns Hopkins Drug Library, for previously undescribed inhibitors of angiogenesis, and one of the most potent hits was identified as the antifungal drug itraconazole (12). We showed that itraconazole inhibited the cell cycle progression of endothelial cells in the G1 phase. Although itraconazole was found to inhibit partially the human lanosterol 14α-demethylase and its knockdown led to a significant, albeit partial, decrease in the proliferation of endothelial cells, the precise molecular mechanism of action of itraconazole has remained unknown. In an attempt to deconvolute the mechanism of inhibition of endothelial cells by itraconazole further, we uncovered a link between intracellular cholesterol trafficking and the mTOR pathway in endothelial cells. Herein, we report that itraconazole causes blockade of cholesterol egress from endosomal/lysosomal compartments to the plasma membrane, which, in turn, leads to inhibition of both mTORC1 and mTORC2. We provide multiple lines of evidence that mTOR activity in endothelial cells requires proper cholesterol trafficking, adding plasma membrane cholesterol to the list of signal inputs to regulate the mTOR pathway.

Results

Given that itraconazole causes cell cycle arrest in the G1 phase, we determined its effects on the expression of a number of known regulators of cell cycle progression at the G1-S transition. No appreciable changes were seen in the levels of CDK2, Cyclin D, and p53; however, the expression of Cyclin A, Cyclin E, and p21 was inhibited by itraconazole in a dose-dependent manner (Fig. 1). Strikingly, the level of p27 was up-regulated, rather than inhibited, by itraconazole (Fig. 1).

Down-regulation of p21 and up-regulation of p27 by itraconazole in HUVECs. HUVECs were treated with itraconazole (ITRA) at the indicated concentrations for 24 h, and cell lysates were subjected to SDS/PAGE followed by Western blot analysis with the indicated antibodies. The relative band intensities of p27 normalized against those of tubulin from the corresponding lanes were provided.

Itraconazole Inhibits Both mTORC1 and mTORC2 in Endothelial Cells.

The opposing changes in the p27 and p21 levels, together with the G1 cell cycle arrest caused by itraconazole, were reminiscent of the effects of rapamycin (13), an inhibitor of mTOR that is also known to inhibit angiogenesis (14). We thus determined the effect of itraconazole on the mTOR pathway in endothelial cells. Like rapamycin, itraconazole inhibited the phosphorylation of p70S6K and 4E-BP1 in a dose-dependent manner (Fig. 2A). In contrast, itraconazole had no effect on the phosphorylation of either ERK or JNK, demonstrating a high level of specificity for the mTOR pathway. In addition to p70S6K and 4E-BP1, which lie downstream of mTORC1, we examined the phosphorylation state of Akt, which lies downstream of mTORC2 (15). Unlike p70S6K, which suffered from a decrease in phosphorylation within half an hour of itraconazole treatment, phosphorylation of Akt at Ser473, which is activated by mTORC2 (15, 16), and Thr308 remained largely unchanged until 8 h after itraconazole treatment (Fig. 2B). On prolonged exposure of endothelial cells to itraconazole, phosphorylation of Akt at both sites was inhibited by the drug in a dose-dependent manner (Fig. 2C). These results suggested that itraconazole inhibited both mTORC1 and mTORC2, and inhibition of mTORC2 likely occurred as a secondary consequence of mTORC1 inhibition.

Itraconazole (ITRA) inhibits both mTORC1 and mTORC2 in HUVECs. (A, C, and D) HUVECs were treated with ITRA and processed in the same manner as in Fig. 1. (B and E) HUVECs were treated with ITRA (2 μM) for the indicated time, and cell lysates were subjected to SDS/PAGE followed by Western blot analysis with the indicated antibodies.

In addition to blocking cell cycle progression in the G1 phase, rapamycin has been shown to induce autophagy in both yeast and mammalian cells (17, 18). We thus determined whether itraconazole was also capable of inducing autophagy in human umbilical vein endothelial cells (HUVECs). Microtubule-associated protein 1 light chain 3 (LC3) is a widely used marker of autophagy. During autophagy, the cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form a conjugate (LC3-II), which can then be recruited to the autophagosome membranes (19). When HUVECs were treated with itraconazole, we observed a dose-dependent and time-dependent induction of LC3-II (Fig. 2 D and E). Using indirect immunofluorescence, we also found that LC3 exhibited a punctate staining pattern indicative of autophagosome formation on treatment of HUVECs with itraconazole (Fig. S1A). Together, these results suggested that the antiangiogenic activity of itraconazole is attributable to its ability to inhibit the mTOR pathway. The different onset of inhibition of mTORC1 and mTORC2 by itraconazole is similar to the effects of rapamycin (20).

Itraconazole Inhibits Cholesterol Trafficking in Endothelial Cells.

The inhibitory effect of itraconazole on endothelial cell proliferation has been shown to be mediated, in part, through its inhibition of lanosterol 14α-demethylase, and hence de novo cholesterol biosynthesis (12). We thus wondered whether the inhibition of the mTOR pathway by itraconazole was also caused by cholesterol deprivation. Cyclodextrin was widely used to directly incorporate cholesterol to cell plasma membranes (21). When cholesterol/cyclodextrin complex was added to HUVECs, it partially reversed the inhibition of endothelial cell proliferation by itraconazole in normal cell growth medium (Fig. 3A), suggesting that itraconazole caused a depletion of cholesterol on the plasma membrane of endothelial cells.

Itraconazole causes accumulation of cholesterol in the late endosome/lysosome, and exogenous cholesterol partially rescues itraconazole inhibition of HUVEC proliferation and mTOR activity. (A) HUVECs were seeded into 96-well plates and incubated with the indicated drugs in the presence and absence of 20 μg/mL cholesterol, cyclodextrin (CD), or cholesterol/CD complex for 24 h. Cells were then pulsed with [3H]-thymidine for 6 h. Cell proliferation rates were normalized to those of cells treated with DMSO, cholesterol, CD, or cholesterol/CD complex only. (B) HUVECs were treated with 2 μM itraconazole for 24 h and then fixed for immunostaining. (C) HUVECs were treated with 2 μM itraconazole in the presence of CD or cholesterol/CD complex for 24 h, and cell lysates were subjected to SDS/PAGE followed by Western blot analyses. The values provided below the bottom of the gel are the relative intensities of the p-S6K1 normalized against those of the S6K1 bands.

To confirm the effect of itraconazole on the cholesterol level in plasma membrane further, we applied filipin, a specific fluorescent marker of unesterized cholesterol (22), to visualize and follow the distribution of free cholesterol in HUVECs in the absence and the presence of itraconazole. In control cells, cholesterol was clearly visible on the plasma membrane as well as on membranes of intracellular organelles such as the nuclear membrane and the ER (Fig. 3B). As expected, there was a dramatic decrease in cholesterol on the plasma membrane under itraconazole treatment. Surprisingly, the majority of cholesterol appeared to be accumulated in an intracellular compartment in the cytosolic space (Fig. 3B). The cholesterol-staining pattern was reminiscent of late endosomes and lysosomes. We thus performed double staining with LAMP-1, a marker of late endosome and lysosome (23), which revealed that free cholesterol was trapped within the late endosomal and lysosomal compartments by itraconazole. This was further confirmed by the lack of colocalization of free cholesterol and protein disulfide isomerase, an ER marker, or cytochrome C, a mitochondria marker (Fig. S1 B and C). These observations suggested that itraconazole inhibited cholesterol trafficking out of the lysosomes and raised the possibility that inhibition of intracellular cholesterol trafficking can lead to inhibition of the mTOR pathway.

To assess the causal relation between inhibition of cholesterol trafficking by itraconazole and inhibition of the mTOR pathway, we treated HUVECs with itraconazole in the presence and absence of cyclodextran-cholesterol and determined the phosphorylation state of p70S6K. As shown in Fig. 3C, inhibition of phosphorylation of p70S6K by itraconazole was partially but significantly reversed by cyclodextran-cholesterol adduct but not by either cholesterol or cyclodextran alone. In addition, the induction of LC3-II and phosphorylation of 4E-BP1 by itraconazole were partially but significantly rescued by the cyclodextran-cholesterol complex (Fig. S2 A and B). In contrast, the inhibition of mTOR by rapamycin in HUVECs was unaffected by the cyclodextran-cholesterol complex (Fig. S2C). These results suggest that disruption of intracellular cholesterol trafficking from the endosome/lysosome to the plasma membrane is largely responsible for inhibition of the mTOR pathway.

Intracellular cholesterol trafficking has been shown to require two cholesterol binding proteins within the lumen of late endosome and lysosome, the Niemann–Pick disease type C (NPC)1 and NPC2 proteins (24
–26). As their names imply, genetic mutations in either gene have been shown to be associated with the corresponding neurological disease in humans and to cause blockade of intracellular cholesterol trafficking (27, 28). To determine whether inhibition of intracellular cholesterol trafficking by alternative means also caused inhibition of the mTOR pathway in endothelial cells, we knocked down NPC1 and NPC2, respectively, using siRNA in HUVECs (Fig. 4A) and determined the effects on the phosphorylation of p70S6K. The siRNA oligonucleotides were identified that caused appreciable, albeit incomplete, down-regulation of NPC1 and NPC2 proteins in HUVECs by 24 h posttransfection. Similar to itraconazole, knockdown of NPC1 or NPC2 also led to the inhibition of p70S6K phosphorylation without significantly affecting the phosphorylation of several other kinases, including PKCα, ERK, and JNK (Fig. 4A and Fig. S2D). In addition to dysregulating the functions of NPC1 or NPC2, two small molecule inhibitors of intracellular cholesterol trafficking are known, U18666A and imipramine (29). Both U18666A and imipramine have been shown to cause accumulation of cholesterol in the late endosome and lysosome. We thus determined their effects on endothelial cell proliferation and mTOR activity using phosphorylation of p70S6K as a readout. Both U18666A and imipramine inhibited HUVEC proliferation in a dose-dependent manner, with IC50 values comparable to those for inhibition of cholesterol trafficking (Fig. S3 A and B). They also exhibited a dose-dependent inhibition of p70S6K phosphorylation (Fig. 4 B and C) with effective concentrations similar to those required for inhibiting intracellular cholesterol trafficking (Fig. S3C). Similar to itraconazole, neither U18666A nor imipramine affected the phosphorylation of PKCα, ERK, or JNK, displaying high specificity for the mTOR signaling pathway (Fig. 4 B and C).

Blockade of cholesterol trafficking by siRNA knockdown of NPC1/2 or the small molecule inhibitors U18666A and imipramine leads to mTOR inhibition. (A) HUVECs were transfected with the indicated siRNA oligonucleotides for 24 h before they were lysed for Western blot analysis. (B and C) HUVECs were treated with the indicated drugs for 24 h before they were harvested for immunoblot analyses.

Among the known activators of the mTOR pathway are insulin and amino acids (30, 31). We thus determined the effects of itraconazole, U18666A, and imipramine on insulin- and amino acid-stimulated mTOR activation in HUVECs. All three inhibitors effectively inhibited the activation of mTOR induced by either insulin or amino acids (Fig. S4 A and B). Thus, proper cholesterol trafficking is necessary for mTOR activation by multiple upstream signaling inputs in endothelial cells. In contrast to the treatment with small molecule inhibitors, however, knockdown of either NPC1 or NPC2 only had marginal effects on mTOR activation by insulin or amino acids (Fig. S4 C and D). It is possible that the knockdown was incomplete and that the remaining NPC1 and NPC2 proteins were sufficient to support mTOR activation by insulin or amino acids.

The mTOR pathway has been shown to play a key role in protein synthesis by regulating cap-dependent translation (30, 32). We wondered whether cholesterol trafficking also regulates protein synthesis in endothelial cells. A luciferase reporter assay was thus performed. As shown in Fig. S5A, pateamine A (PatA), a known inhibitor of eukaryotic translation initiation, dramatically inhibited cap-dependent translation. Somewhat surprisingly, rapamycin had a much smaller effect on cap-dependent translation than PatA, which suggested that the dependence of cap-dependent translation on mTOR is not as pronounced in endothelial cells. The extent of inhibition of cap-dependent inhibition by rapamycin is in agreement with that observed previously (33). Nevertheless, itraconazole also had a small but significant effect on cap-dependent translation similar to rapamycin. Furthermore, knockdown of either NPC1 or NPC2 had a similar effect on cap-dependent translation (Fig. S5B). The similar effects of itraconazole, rapamycin, and NPC1/2 knockdown on cap-dependent translation in endothelial cells are consistent with their common inhibition of mTOR.

Reversal of Itraconazole-Induced Accumulation of Cholesterol in Late Endosomes/Lysosomes and Inhibition of mTOR by Thapsigargin.

The NPC phenotype has recently been shown to be attributable to the dysregulation of endosomal/lysosomal calcium homeostasis, and thapsigargin, which releases ER calcium, was shown to correct the cellular NPC phenotype (34). We thus examined the effect of thapsigargin on cholesterol accumulation in endosomes/lysosomes caused by itraconazole. Thapsigargin restored the transport of cholesterol from endosomes/lysosomes to the plasma membrane in HUVECs (Fig. 5A). We next determined whether thapsigargin treatment could reverse the inhibition of the mTOR pathway by itraconazole. Indeed, thapsigargin not only reversed inhibition of p70S6K phosphorylation by itraconazole but conferred resistance to both U18666A and imipramine (Fig. 5B), indicating that restoration of cholesterol trafficking by thapsigargin confers resistance to mTOR inhibition by all three inhibitors of intracellular cholesterol transport. In contrast, inhibition of mTOR by rapamycin, which does not affect intracellular cholesterol trafficking, could not be reversed by thapsigargin (Fig. S6A). In addition to its effect on ER calcium release, thapsigargin is known to induce unfolded protein response (35). We thus determined the ability of tunicamycin, another known inducer of unfolded protein response, to reverse the inhibition of the mTOR pathway by itraconazole, U18666A, and imipramine. Unlike thapsingargin, tunicamycin is incapable of reversing the inhibition of p70S6K phosphorylation by any of the three inhibitors (Fig. S6B). Together, these results indicated that proper cholesterol trafficking is required for mTOR signaling in endothelial cells.

Effects of thapsigargin (TG) and cholesterol/cyclodextrin complex on the inhibition of cholesterol trafficking and mTOR activity by itraconazole (ITRA), U18666A, and imipramine. (A and B) HUVECs were preincubated with 1 μM TG for 1 h before they were treated with 2 μM itraconazole, 30 μM imipramine, or 10 μM U18666A for an additional 24 h. They were then fixed for filipin staining (A) or harvested for immunoblot analysis (B). (C) HUVECs were incubated with 10 μM U18666A or 30 μM imipramine in the presence or absence of 4 μg/mL cholesterol/0.1% cyclodextrin complex for 24 h before being harvested for immunoblot analyses using the indicated antibodies.

In addition to thapsigargin, we determined the effect of exogenously administered cholesterol/cyclodextrin complex on the inhibition of mTOR by U18666A and imipramine. Similar to what was observed for itraconazole, the inhibition of mTOR by either U18666A or imipramine was significantly reduced in the presence of cholesterol/cyclodextrin, consistent with the notion that the inhibitory effects of those two compounds arose from their effects on cholesterol transport from the lysosome to the plasma membrane (Fig. 5C).

Discussion

Itraconazole is a widely used antifungal drug. We recently reported that in addition to its antifungal activity, itraconazole possesses potent antiangiogenic activity both in vitro and in vivo (12). In the course of delineating its antiangiogenic mechanism, we found that itraconazole specifically inhibited the mTOR pathway in endothelial cells. It is well known that mTOR plays an essential role in angiogenesis. Rapamycin and its analogue, temsirolimus, both inhibit tumor angiogenesis in vivo and directly inhibit endothelial cell proliferation in vitro (14, 36). mTOR exists in two complexes in cells: mTORC1 and mTORC2, both of which are required for endothelial cell proliferation. In pathological angiogenesis, mTORC1 mediates an earlier and transient response, whereas mTORC2’s effect is more sustained (37). In endothelial cells, rapamycin could not only inhibit mTORC1 but inhibit mTORC2 after long incubation because of its disruption of mTORC2 assembly (20). The dual inhibition of both mTORC1 and mTORC2 was thought to be the key to rapamycin's antiangiogenic activity (33). In this study, we found that itraconazole, like rapamycin, also inhibited both mTORC1 and mTORC2 in endothelial cells on prolonged exposure. The dual inhibition of both mTORC complexes by itraconazole offers a plausible explanation for the potent cell cycle effect of itraconazole on endothelial cells.

Further mechanistic deconvolution revealed that itraconazole interferes with cholesterol trafficking and causes an apparent NPC phenotype in endothelial cells. Whether the blockade of cholesterol trafficking through the late endosome and lysosome is solely responsible for the depletion of cholesterol in the plasma membrane and the accompanying inhibition of mTOR activity, however, was not immediately clear, because itraconazole has been shown to inhibit de novo cholesterol biosynthesis, which could also lead to depletion of cholesterol in the plasma membrane. To distinguish between the two alternative effects of itraconazole as the potential cause of mTOR inhibition, we also determined the effects of itraconazole on its putative target, human lanosterol 14α-demethylase, by measuring the cellular concentrations of lanosterol. Indeed, itraconazole caused an accumulation of lanosterol when HUVECs were cultured in the presence of mevalonate (Table S1). However, lanosterol became undetectable when exogenous mevalonate was left out of the growth medium, suggesting that the de novo cholesterol biosynthetic pathway was inactive. It remains to be determined whether the negative feedback inhibition of the biosynthetic pathway was caused by the accumulation of an undetectable level of lanosterol, by the accumulation of cholesterol in the late endosome and lysosome, or by a combination of both. It is possible that the cholesterol depletion in the plasma membrane by itraconazole is caused by a dual mechanism: inhibition of de novo biosynthesis and trafficking through the lysosome.

Building on the preliminary observations with itraconazole, we employed other known inhibitors of cholesterol trafficking, U18666A and imipramine, and demonstrated that inhibition of cholesterol trafficking alone is sufficient to inhibit mTOR activity. It is worth noting that U18666A, similar to itraconazole, has also been reported to inhibit cholesterol biosynthesis (38). Unlike U18666A and itraconazole, however, imipramine has no effect on cholesterol biosynthesis, and its cellular effects can only be attributed to blockade of cholesterol trafficking. That imipramine also caused inhibition of mTOR activity indicates that inhibition of intracellular cholesterol trafficking alone is sufficient to block the mTOR pathway. This conclusion was further supported by the demonstration that knockdown of NPC1 or NPC2, known mediators of cholesterol transport through the late endosome and lysosome, also inhibited mTOR activity in HUVECs.

Our findings implicate cholesterol homeostasis as a potential signal input for the mTOR pathway. Thus, in the absence of proper cholesterol distribution to the plasma membrane (and likely other intracellular membranes), the mTOR pathway is turned off, leading to the inhibition of endothelial cell proliferation. It is apparent that the defect in mTOR was caused, in part, by the depletion of cholesterol in the plasma membrane. However, it is unclear whether there exists a cellular sensing mechanism for membrane cholesterol levels, and if so, whether it is mediated through a specific receptor. It is formally possible that the depletion of cholesterol from the plasma membrane could cause a global defect in either nutrient transport and/or dysfunction of certain cell surface receptors required for endothelial cell growth.

We have shown that activation of mTOR by such upstream signals as insulin and amino acids is sensitive to inhibition by itraconazole, suggesting that the site of inhibition lies somewhere in the common pathway downstream of both insulin and amino acid signaling. Among the more proximal upstream activators of mTOR are Rag guanosine triphosphatases (GTPases), which have recently been reported as regulators of mTORC1 (39). We overexpressed constitutively active Rag GTPases to activate mTOR in the presence of itraconazole. Interestingly, itraconazole also inhibited Rag-mediated mTOR activation (Fig. S7A). Moreover, we also examined the effect of itraconazole on the subcellular distribution of mTOR and found it to be unaffected by itraconazole (Fig. S7B). Further experiments will be needed to identify the signaling protein in the mTOR pathway that cross-talks with cholesterol trafficking.

We have previously shown that itraconazole is selective for endothelial cells, suggesting that endothelial cells may be particularly sensitive to perturbation of cholesterol homeostasis. For comparison, we also determined the effect of itraconazole on the proliferation and mTOR activity in 293T and HeLa cells. We have previously shown that HeLa cells are significantly less sensitive to itraconazole than HUVECs (12). Similarly, 293T cells are also significantly less sensitive than HUVECs to the drug; approximately 25% of cells remained resistant to the highest concentrations of itraconazole applied (Fig. S8A). In agreement with the cell proliferation data, the mTOR activity in both 293T and HeLa cell lines, as measured by p70S6K phosphorylation, is also less sensitive to inhibition by itraconazole (Fig. S8 B and C) than that in HUVECs (Fig. 2A). Similar to itraconazole, knockdown of either NPC1 or NPC2 in 293T cells had little effect on p70S6K phosphorylation (Fig. S8D), further underscoring the unique sensitivity of the mTOR pathway in endothelial cells to perturbation of cholesterol trafficking. Why do endothelial cells exhibit unique high sensitivity to inhibitors of intracellular cholesterol transport? It is tempting to speculate that endothelial cells may have evolved a higher level of cholesterol intake from the plasma they are naturally exposed to, and thus greater dependence on transport of cholesterol from endosomes/lysosomes. The essential role of cholesterol homeostasis in the activity of the mTOR pathway in endothelial cells suggests that the intracellular cholesterol trafficking pathway, including NPC1 and NPC2, may serve as a promising target for developing inhibitors of angiogenesis, as exemplified by the unique antiangiogenic activity of itraconazole.

Materials and Methods

Materials.

HUVECs and medium were purchased from Lonza, Inc. Cells were cultured in endothelial cell growth medium (EGM)-2 media at 5% CO2. 293T cells were cultured in DMEM/10% FBS at 5% CO2 (vol/vol). Antibodies for Cdk2, Cyclin A, Cyclin D, Cyclin E, p21, p27, p53, tubulin, Akt, p-JNK, S6K, NPC2, protein disulfide isomerase, and cytochrome C were purchased from Santa Cruz, Inc. Antibodies for p-S6K (thr389), p-4E-BP1 (ser65), p-Akt (thr308), p-Akt (ser473), and p-ERK were purchased from Cell Signaling. Antibody for NPC1 was purchased from Proteintech. Antibody for p-pkcα was purchased from Millipore. Antibody for LC3b was purchased from Abcam. LAMP-1 antibody developed by J.T. August and James Hildreth was obtained from the Developmental Studies Hybridoma Bank under the auspices of the National Institute of Child Health and Human Development (NICHD) and maintained by the University of Iowa, Department of Biology. siRNA oligos for NPC1 (SI00005887) and NPC2 (SI03026632, SI03093951) and control oligos were purchased from Qiagen. Cholesterol, filipin, and (2-hydroxypropyl)-β-cyclodextrin were purchased from Sigma.

Filipin Staining.

Cells were fixed with 4% (wt/vol) paraformaldehyde in PBS for 30 min and stained with 50 μg/mL filipin in PBS at room temperature for 2 h. Cells were then washed with PBS three times and mounted. Images were captured using a Zeiss LSM510 confocal microscope.

Cholesterol Rescue Assay.

The cholesterol/cyclodextrin complexes were prepared as described previously (21). Cells were treated with cholesterol/cyclodextrin complexes along with the indicated drugs for 24 h before they were used for immunoblotting or cell proliferation assay.

Acknowledgments

We are grateful to Dr. Peter Espenshade and Ms. Clara Bien for technical assistance and Drs. Kun-Liang Guan and Ta-Yuan Chang for provision of Rag plasmids and cell lines. We thank Benjamin Nacev and Joong Sup Shim for critical comments on the manuscript. The financial support from the National Cancer Institute, Flight Attendant Medical Research Institute, The Clinical and Translational Science Award, Keck Foundation, Patrick C. Walsh Prostate Cancer Research Fund, and Commonwealth Foundation is gratefully acknowledged.

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